Metabolic rewiring coupled to the production of reactive oxygen and nitrogen species (RONS)

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Project Summary/Abstract Free radicals are more than toxic by-products of metabolism. Many cells produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) using specialized enzymes, NADPH oxidases (NOXs) and nitric oxide synthases (NOSs) respectively, for specific functions. For instance, innate immune cells produce RONS in large amounts during inflammation, and endothelial cells and neurons can produce them for signaling. RONS production is an important part of many physiological processes as well as numerous diseases, including inflammatory disorders, cardiovascular diseases, neurodegeneration, etc. We propose to investigate how NOX- and NOS-dependent RONS production is coupled to substantial remodeling of cellular metabolism. Specifically, we will address two key questions: (1) How is cellular metabolism remodeled to enable RONS production? Both NOXs and NOSs require NADPH as the electron donor to drive the reactions. Thus, activation of NOX or NOS greatly increases cellular NADPH demand and can significantly lower NADPH/NADP+ redox ratio. We recently discovered that in activated neutrophils, NOX activation is quantitatively coupled to the metabolic shift from glycolysis to cyclic pentose phosphate pathway (a unique mode for glucose metabolism with ultra-high NADPH yield) to meet this demand. We propose to investigate the molecular mechanism allowing neutrophils to complete such substantial metabolic switch in a very short time, which is essential for them to immediately mount the first line of innate immune defense. We will next reveal the metabolic strategies other cells use to power up RONS production using approaches including isotopic-tracing based metabolic flux analysis and genetic manipulation. (2) How does the increase in RNS regulates cellular metabolism? It is well known that RONS can impact cell functions through protein cysteine oxidation or nitrosylation. Intriguingly, we recently found that the inducible RNS production in activated macrophages drives dynamic metabolic remodeling via a novel mechanism: RNS specifically and efficiently inactivate two crucial mitochondrial enzymes, pyruvate dehydrogenase and oxoglutarate dehydrogenase, via covalent modifications of the catalytic lipoic arm in their E2 subunits. We seek to understand of the biochemical mechanisms for such RNS-driven regulation in-depth, specifically, (i) elucidate the role of coenzyme A, the thiol-containing substrate for these enzymes, in delivering NO- mediated modifications specifically onto the catalytic lipoic moiety; (ii) examine the mechanistic link between such lipoic modifications with the cysteine nitrosylation on their E3 subunit; (iii) examine the reversibility of such RNS- mediated modifications of lipoic arm and the mechanism driving the recovery. We will further investigate how this mechanism regulates other lipoic arm dependent- enzymes, and elucidate the downstream effects of such regulation in cellular physiology in various RNS-producing cells (e.g. regulation of pyruvate dehydrogenase by NO can modulate histone acetylation and downstream cell functions by changing acetyl-coA availability).